We are using a yeast system we constructed earlier that allows us to evaluate function of any Hsp70 isoform in yeast. We are using this system to investigate Hsp70 homologs within and across species with regard to cell growth and propagation of different prions. This very sensitive system gives us the unique ability to distinguish exquisite functional differences among nearly identical Hsp70 isoforms and provides a means to approach the problem of uncovering the underlying mechanisms. The constitutively expressed S. cerevisiae Hsp70 isoforms Ssa1 and Ssa2 are 98% identical and their stress-inducible counterparts Ssa3 and Ssa4 share 88% identity and are 80% identical to Ssa1/2. Using our system we have identified structural differences between Ssa1 and Ssa2 that underlie the differences in the way these isoforms function in both prion propagation and protein degradation. Our findings provide insight into how nearly identical Hsp70 isoforms can have distinct activities with regard to their roles in important cellular processes, and in the replication and growth processes necessary for propagation of yeast prions. Our work also reveals how functionally redundant proteins can still perform specialized tasks in the cell. Current work focuses on understanding how the structural differences influence enzymatic activities at a molecular level, which could lead to design of strategies targeting Hsp70 function in vivo in ways that would hinder amyloid accumulation. We are also using our system to investigate to what degree the structural differences contribute to differences in intrinsic Hsp70 activities or interactions with co-chaperones that regulate Hsp70.